This invention is directed to combinatorial chemistry libraries containing fused 2,4-pyrimidinediones. This invention is further directed to methods for constructing combinatorial chemistry libraries containing fused 2,4-pyrimidinediones. This invention is still further directed to methods for the identification of bioactive fused 2,4-pyrimidinediones.
Modem day drug discovery is a multi-faceted endeavor. Researchers commonly delineate a biochemical pathway that is operative in a targeted pathological process. This pathway is analyzed with an eye toward determining its crucial elements: those enzymes or receptors that, if modulated, could inhibit the pathological process. An assay is constructed such that the ability of the important enzyme or receptor to function can be measured. The assay is then performed in the presence of a variety of molecules. If one of the assayed molecules modulates the enzyme or receptor in a desirable fashion, this molecule may be used directly in a pharmaceutical preparation or can be chemically modified in an attempt to augment its beneficial activity. The modified molecule that exhibits the best profile of beneficial activity may ultimately be formulated as a drug for the treatment of the targeted pathological process.
With the use of high-throughput screening techniques, one can assay the activity of tens of thousands of molecules per week. Where molecules can only be synthesized one at a time, the rate of molecule submission to an assay becomes a debilitating, limiting factor. This problem has led researchers to develop methods by which large numbers of molecules possessing diverse chemical structures can be rapidly and efficiently synthesized. One such method is the construction of chemical combinatorial libraries.
Chemical combinatorial libraries are diverse collections of molecular compounds. Gordon et al. (1995) Acc. Chem. Res. 29:144-154. These compounds are formed using a multistep synthetic route, wherein a series of different chemical modules can be inserted at any particular step in the route. By performing the synthetic route multiple times in parallel, each possible permutation of the chemical modules can be constructed. The result is the rapid synthesis of hundreds, thousands, or even millions of different structures within a chemical class.
For several reasons the initial work in combinatorial library construction focused on peptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res. 37:487-493; Houghton et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Fodor et al. (1991) Science 25:767. The rapid synthesis of discrete chemical entities is enhanced where the need to purify synthetic intermediates is minimized or eliminated; synthesis on a solid support serves this function. Construction of peptides on a solid support is well-known and well-documented. Obtaining a large number of structurally diverse molecules through combinatorial synthesis is furthered where many different chemical modules are readily available; hundreds of amino acid modules are commercially available. Finally, many peptides are biologically active, making them interesting as a class to the pharmaceutical industry.
The scope of combinatorial chemistry libraries has recently been expanded beyond peptide synthesis. Polycarbamate and N-substituted glycine libraries have been synthesized in an attempt to produce libraries containing chemical entities that are similar to peptides in structure, but possess enhanced proteolytic stability, absorption and pharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371. Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine libraries have been synthesized, expanding combinatorial chemistry to include heterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc. 114:10997-10998; Murphy et al. (1995) J. Am. Chem. Soc. 117:7029-7030; and Gordon et al. (1995) Biorg. Medicinal Chem. Lett. 5:47-50.
Pyrimidinediones are a class of bioactive, heterocyclic molecules that have attracted considerable attention in the pharmaceutical industry. The benzo derivatives of this series (2,4-quinazolinediones) are represented as anti-inflammatory agents, analgesics, anticonvulsants, CNS agents, serotonin uptake inhibitors, antihypertensive agents, cardiovascular agents, and fungicides. Maillard et al. (1968) Fr. Chim. Ther. 3:100-106; Montginoul et al. (1988) Ann. Pharm. Franc. 46:223-232; Lowe et al. (1991) J. Med. Chem. 34:624-628; Mignani et al. (1993) Bioorg. Med. Chem. Lett. 3:1913-1918; Can. Pat. Appl. CA 2053475; Eur. Pat. Appl. EP 481342; and Smith et al. (1996) Bioorg. Med. Chem. Lett. 6:1483-1486. Naturally occurring bioactive compounds, such as theophylline and theobromine, that have found application as cardiotonic agents, broncholytic agents, vasodilators, psychotonic agents, and circulation analeptic agents are heterocyclic ring fused 2-4-pyrimidinediones. Roth et al. In: Pharmaceutical Chemistry. Volume 1: Drug Synthesis (1988) John Wiley and Sons. Recently, synthetic heterocyclic fused 2,4-pyrimidinediones, such as thieno and furopyrimidine-2,4-diones have been patented as serotonin antagonists and alpha adrenergic blocking agents. U.S. Pat. No. 4,835,157.
Methods for the solution phase preparation of fused 2,4-pyrimidinediones have been reported. For example, the solution phase synthesis of pyridopyrimidinediones and quinazolinediones by the reaction, respectively, of an aminonicotinic ester or anthranilate derivative with isocyanate has been described. Lowe et al. (1991) J. Med. Chem. 34:624-628. The reaction of a carbomethoxyphenyl isocyanate with an amino acid is a further described example of solution phase chemistry used to construct fused pyrimidinediones. Canonne et al. (1993) Heterocycles 36:1305-1314. Little work, however, has been reported on the solid phase synthesis of fused 2,4-pyrimidinediones.
An 11 step synthesis of particularly substituted 2,4-quinazolinediones using solid phase synthesis that proceeds through an anthranilate intermediate has been described. Buckman et al. (1996) Tetrahedron Lett. 37:4439-4442. This synthetic route is inherently limited, however, to the production of phenolic 2,4-quinazolinediones due to the mode of connection between the 2,4-quinazolinedione and the solid support. Furthermore, the synthetic route is limited by the harshness of the reaction conditions employed. For instance, the cyclization of the pyrimidinedione ring requires the presence of strong potassium hydroxide: a reagent that could cause cleavage of the compound from the solid support, destroy certain functional groups such as esters, or racemize chiral groups such as amino acid derivatives. Finally, the synthetic route requires the use of air-sensitive reagents, such as lithium benzyloxazolidinone, making the automation of the synthetic protocol difficult, thus potentially reducing its application to the manual synthesis of a limited number of 2,4-quinazolinediones.
A 5 step synthesis of 2,4-quinazolinediones using solid phase synthesis that proceeds through an anthranilate intermediate has been described. Smith et al. (1996) Biorg. and Medicinal Chem. Lett. 6:1483-1486. This route is limited in that it does not allow for the production of 2,4-quinazolinediones bound to a solid support: the last step of the synthesis both completes the pyrimidinedione ring and releases the formed compound from the solid support. The route is further limited in that it employs a moisture-sensitive chloroformate derivatized polymeric support that has to be prepared immediately before use. Finally, the final synthetic step requires a high reaction temperature virtually excluding the application of standard equipment used for the automated synthesis of combinatorial libraries. Therefore the scope of the application of this method is severely limited.
A four step synthesis of pyrido[2,3-d]pyrimidines has been described. Gordeev et al. (1996) Tetrahedron Lett. 37:4643-4646. This route is limited to the production of particularly substituted pyridopyrimidines. Due to the nature of the linkage between the pyridopyrimidine and the solid support, only those compounds with a carboxyl group in the 6-position can be accessed.
Methods for nucleophilic aromatic substitution reactions have been described, where a fluoride substituent on an aromatic ring is displaced from a chemical intermediate that is attached to a solid support. MacDonald et al. discloses the use of a nucleophilic aromatic substitution reaction on a solid support as a step in the preparation of a quinolone library. (1996) Tetrahedron Lett. 37: 4815-4818. Phillips et al. describes the use of a nucleophilic aromatic substitution reaction in the solid phase preparation of benzimidazoles. (1996) Tetrahedron Lett. 37: 4887-4890. Shapiro et al. delineates the use of 19F NMR monitoring of a nucleophilic aromatic substitution reaction on a solid support. (1996) Tetrahedron Lett. 37: 4671-4674. None of these references either disclose or suggest the use of a nucleophilic aromatic substitution reaction in the preparation of a fused 2,4-pyrimidinedione library on a solid support.
The cited references in the background section, and in the following sections, are herein incorporated by reference.
The present invention provides a combinatorial library that contains a fused 2,4-pyrimidinedione (shown below, I): 
where the xe2x80x98Axe2x80x99 ring is an aromatic ring, a heteroaromatic ring, an aliphatic ring or substituted versions thereof.
In one embodiment, the combinatorial library contains fused 2,4-pyrimidinediones selected from the group: 2,4-quinazolinediones, pyrimidopyrimidinediones, pyridopyrimidinediones, 2,4-pteridinediones, and azolopyrimidinediones (FIG. 1, where R2-R6 can independently be alkyl, aryl, heteroaryl, electron withdrawing groups, and amino acid derivatives). Preferably, the combinatorial library contains fused 2,4-pyrimidinediones including but not limited to: 2,4-quinazolinediones, pyrimidopyrimidinediones, and azolopyrimidinediones.
In another embodiment, the combinatorial library contains fused 2,4-pyrimidinediones, where R1 in the fused 2,4-pyrimidinedione is: 
where R7, R8, and R9 are independently selected from the group H, alkyl, aryl, OH, NH2, NHR10, NR10R11, SH, SR10, xe2x95x90O, C(O)R10, and an amino acid side chain; and where Y is selected from the group alkyl, aryl, O, NH, and NR10; and where XH is selected from the group CO2H, CO2R10, C(O)R10, SH, OH, NH2, NHR10, C(O)NHOH, and C(O)NHR10; and further where m, n, o, and p vary independently from 0 to 4, and where R10 and R11 are independently selected from the group alkyl, aryl and heteroaryl.
In another embodiment, the combinatorial library contains fused 2,4-pyrimidinediones, where R1 in the fused 2,4-pyrimidinedione is: 
where R7 and R8 are independently selected from the group H, alkyl, aryl, OH, NH2, NHR10, NR10R11, SH, SR10, xe2x95x90O, C(O)R10, and an amino acid side chain; and where m, n, and o vary independently from 0 to 4; and where R10 and R11 are independently selected from the group alkyl, aryl and heteroaryl.
In another embodiment, the combinatorial library contains fused 2,4-pyrimidinediones, where R1 in the fused 2,4-pyrimidinedione is: 
where R7 is independently selected from the group H, alkyl, aryl, OH, NH2, NHR10, NR10R11, SH, C(O)R10, and an amino acid side chain; and where XH is selected from the group CO2H, CO2R10, C(O)R10, SH, OH, NH2, NHR10, C(O)NHOH, and C(O)NHR10; and where m, and n vary independently from 0 to 4; and where R10 and R11 are independently selected from the group alkyl, aryl and heteroaryl.
The present invention also provides a combinatorial library that contains fused 2,4-pyrimidinediones, where the fused 2,4-pyrimidinediones are connected to a solid support via R1,and where the fused 2,4-pyrimidinedione is selected from the group: 2,4-pteridinediones, pyridopyrimidinediones, pyrimidopyridazinediones and azolopyrimidinediones.
In one embodiment, the combinatorial library contains fused 2,4-pyrimidinediones selected from the group: 2,4-pteridinediones, pyridopyrimidinediones, and pyrimidopyridazinediones.
In another embodiment, the combinatorial library contains pyridopyrimidines.
The present invention also provides a combinatorial library that contains fused 2,4-pyrimidinediones, where the fused 2,4-pyrimidinediones are connected to a solid support through a substituent at the 3-position.
In another embodiment, the combinatorial library contains fused 2,4-pyrimidinediones selected from the group: 2,4-quinazolinediones, pyrimidopyrimidinediones, pyridopyrimidinediones, 2,4-pteridinediones, and azolopyrimidinediones.
In another embodiment, the combinatorial library contains fused 2,4-pyrimidinediones selected for the group: 2,4-quinazolinediones, pyrimidopyrimidinediones, and pyridopyrimidinediones.
In another embodiment, the combinatorial library contains a 2,4-quinazolinedione.
The present invention also provides a combinatorial library that contains fused 2,4-pyrimidinediones that is prepared by cleaving the fused 2,4-pyrimidinediones from a solid support.
The present invention also provides a combinatorial library that contains fused 2,4-pyrimidinediones that possess a substituent at the 3-position, where the library is prepared by cleaving the fused 2,4-pyrimidinedione from a solid support.
In one embodiment, this method includes a step where a fluoride substituent is displaced in a nucleophilic aromatic substitution reaction.
The present invention also provides a method of producing a combinatorial library that contains fused 2,4-pyrimidinediones, where the fused 2,4-pyrimidinediones are connected to a solid support, and where the fused 2,4-pyrimidinedione is selected from the group: 2,4-pteridinediones, pyridopyrimidinediones, pyrimidopyridazinediones and azolopyrimidinediones.
In one embodiment, this method includes a step where a fluoride substituent is displaced in a nucleophilic aromatic substitution reaction.
The present invention also provides a method of producing a combinatorial library that contains fused 2,4-pyrimidinediones, where the fused 2,4-pyrimidinediones are connected to a solid support through a substituent at the 3-position.
In one embodiment, this method includes a step where a fluoride substituent is displaced in a nucleophilic aromatic substitution reaction.
The present invention also provides a method of screening a library that contains a fused 2,4-pyrimidinedione.
In one embodiment, the fused 2,4-pyrimidinediones that are screened are connected to a solid support.
In another embodiment, the fused 2,4-pyrimidinediones that are screened are in solution.